We apply for the first competitive renewal of ROl A141420 'Second receptors for primate immunodeficiency viruses." Our overall goal is to gain additional knowledge of the roles played by coreceptors in mediating HIV-1 entry, with emphasis on understanding how inhibitors of this process act, where they bind, and how the virus can escape their actions. In Specific Aim 1, we will characterize escape pathways for coreceptor-targeted inhibitors of H!V-1 entry in vitro. We will generate escape mutants of an R5 HIV-1 primary isolate, using primary cells as targets, under the selection pressure of small molecule inhibitors, specific MAbs and CC-chemokines. We will determine whether the escape mutant viruses still use CCR5 or have instead, or as well, evolved the ability to use other coreceptors such as CXCR4. We will determine which sequence changes in the env gene are responsible for conferring the resistant phenotype. If the resistant viruses still use CCR5, we will investigate how, by using specific point mutants of CCR5. In Specific Aim 2, we will evaluate HIV-1 entry inhibitors using cells derived from the gut and from cord blood, in macrophages and in cells expressing different levels of CCR5 We will see whether the potency of CCR5 inhibitors is influenced by the level of CCR5 expression on the surface of CD4+ target cells, in vitro, using both primary cells and engineered cell lines. We will determine whether either parameter correlates with known CCR5 promoter genotypes, since this may influence cell surface CCR5 expression. We will study relevant target cells for HlV-1 replication in vivo, including cells isolated from gut associated lymphoid tissue. We will also study the mechanism of action of CCR5 inhibitors, using biosensor methodology. Specific Aim 3 is to investigate the determinants of rapid progression in SIV-infected macaques and thereby identify the SIV coreceptor that is the counterpart of CXCR4 in HIV-1 -infected humans. We will use specific inhibitors targeted at CCR5 to block this pathway for SIV entry into macaque PBMC, and thereby identify donor macaques that exhibit CCR5-inhibitor insensitive SIV entry in vitro. We hypothesize that this is due to the use of a different SIV coreceptor that is expressed at sufficient levels in cells from only a subset of animals. We will determine whether there is any correlation between CCR5-inhibitor insensitive SIV entry in vitro, and rapid progression upon SIV infection in vivo, by performing retrospective analyses of the outcome of Sly infection. We will identify what coreceptor(s) mediate CCR5-inhibitor insensitive SIV entry in vitro.